Reference Profiles of Extracellular RNA in 4 Body Fluids of Healthy Humans

描述（由申请人提供）： 在已知的血液分子中，有一类新的细胞外RNA（exRNA）。其中一些是优秀的生物标志物候选者，但也有大量的exRNA来自微生物组。内源性和外源性exRNA都携带着大量的信息，可能是细胞功能的效应子。美国国立卫生研究院共同基金exRNA联盟目前正在研究和应用exRNA在人类的诊断和治疗。这项工作的基础，在健康个体的exRNA谱的可靠配置文件，是必要的。我们组建了一支经验丰富的跨学科团队，仔细表征这些参考RNA谱，并改进方法。PI将与Co-PI的Debbie Nickerson密切合作（UW基因组学中心共同主任，测序）和王凯（系统生物学研究所，数据分析），共同研究者，Elaine Peskind，VA中心和UW，选择和提供样本），以及共同研究者提供建议，咨询和合作（Paul Wilmes，卢森堡大学; Aleks Milosavljevic，Baylor;和Muneesh Tewari，密歇根大学）我们建议从相同的健康受试者中产生，分析和传播血浆，血清，CSF和唾液的exRNA谱：三个年龄组（31至101岁）中大致相同数量的男性和女性。这些同时从相同受试者采集的血清、血浆、唾液和CSF的匹配样本是本提案的关键资产。我们最近的工作揭示了exRNA研究的一个新维度。血浆中微生物RNA（细菌和真菌）的存在提出了关于其起源和功能的重要问题。我们将使用我们的计算管道，包括来自DMRR（DIAC）的工具，来表征脂质囊泡（人血清，血浆，唾液和CSF）内外的内源性exRNA（en-exRNA）和外源性exRNA（ex-exRNA）。我们将为每个样品制作四个NextGen文库：囊泡内和囊泡外，以及短和长插入文库。我们建议在五年内描述一组180名受试者的特征，并在ECRP联盟的DMRR创建和维护的exRNA图谱中分析和存款配置文件数据和元数据。我们将完成一种新方法的开发和测试，以产生序列特异性文库偏倚校正因子。由于微生物序列数据库更新，我们将定期重新表征ex-exRNA。所产生的数据将使我们能够检查个体exRNA和exRNA簇的差异作为受试者属性（包括年龄和性别）的函数。该项目将分两个阶段进行。在第一阶段（第1年和第2年），将在数据采集和分析期间测试各种方法，包括偏差校正方法。第二阶段（第3-5年）将仅包括数据生成和分析。我们提案的主要重点和优势是严格收集、匹配相同受试者的多种体液样本，重点是准确获取和分析exRNA，以及我们在拟议工作的各个方面的丰富经验。

DESCRIPTION (provided by applicant): Among the known blood molecules is a new class, the extracellular RNAs (exRNAs.) Some of these are excellent biomarker candidates, but there are also a significant number of exRNAs derived from the microbiome. Both endogenous and exogenous exRNAs carry much information and could be effectors of cellular function. The NIH Common Fund exRNA consortium is now researching and applying exRNA in humans to diagnostics and therapeutics. A foundation for this effort, reliable profiles of the exRNA spectrum in healthy individuals, is needed. We have assembled an experienced, interdisciplinary team to carefully characterize these reference RNA profiles, and to improve the methods for doing so. The PI will work closely with Co-PI's Debbie Nickerson (UW Genomics Center co-Director, Sequencing), and Kai Wang (Institute for Systems Biology, data analysis), Co-Investigator, Elaine Peskind, VA Center and UW, selecting and providing samples), and co-investigators to provide advice, consultation, and collaboration (Paul Wilmes, Univ. of Luxembourg; Aleks Milosavljevic, Baylor; and Muneesh Tewari, Univ. of Mich.) We propose the generation, analysis, and dissemination of exRNA profiles of plasma, serum, CSF and saliva from the same healthy subjects: roughly equal numbers of men and women in three age categories (31 to 101 years.) These matched samples of serum, plasma, saliva and CSF collected at the same time from the same subjects are a key asset of this proposal. A new dimension in exRNA research was revealed by our recent work. The presence of microbial RNA (bacterial and fungal) in blood plasma raises important questions concerning its origins and functions. We will use our computational pipeline, including tools from the DMRR (DIAC), to characterize both endogenous exRNAs (en-exRNA) and exogenous exRNA (ex-exRNA) in and outside of lipid vesicles (in human serum, plasma, saliva and CSF.) We will do four NextGen libraries per sample: in and out of vesicles, and short and long insert libraries. We propose to characterize a set of 180 subjects over five years, and analyze and deposit profile data and metadata in the exRNA Atlas created and maintained by the DMRR of the ECRP consortium. We will complete the development and testing of a novel approach to generate sequence-specific library bias correction factors. We will periodically re-characterize the ex-exRNA due to microbial sequence database updates. The data generated will enable us to examine the variance in individual exRNAs and clusters of exRNAs as a function of subject attributes (including age and gender.) The project will be carried out in two phases. In the first phase (years 1 and 2), methods will be tested during data acquisition and analysis, including the bias correction method. The second phase (years 3-5) will consist of data generation and analysis only. A major emphasis and strength of our proposal is the rigorously collected, matched samples of multiple body fluids on the same subjects, with emphasis on accurate acquisition and analysis of exRNA and our extensive experience in all aspects of the proposed work.